Project Summary Antibiotic resistance in bacterial pathogens is quickly rendering current antibiotics obsolete; thus, there is a need to discover additional molecular targets and develop novel strategies for treating bacterial infections. We have previously determined that the important pathogen and powerful model Listeria monocytogenes (Lmo) is rendered sensitive to cell wall stress in vitro, including that imposed by ?-lactam antibiotics and host-derived defenses such as lysozyme, upon genetic disruption or pharmacological inhibition of PrkA, a penicillin-binding protein and serine/threonine-associated (PASTA) kinase. PASTA kinases are activated by disturbances in cell wall homeostasis and mediate a stress response through largely unidentified phosphotargets and downstream pathways. PrkA is also required for cytosolic survival of Lmo, suggesting Lmo faces cell wall stress in the host cell cytosol. While the host cytosol is known to be restrictive to non-adapted bacteria, the cell autonomous defenses (CADs) responsible for killing bacteria remain largely unidentified. In this proposal, we will test the hypothesis that PrkA phosphosubstrates mediate cell wall stress responses important for cytosolic survival and virulence using an orthogonal approach combining genetic and phosphoproteomic analyses. Furthermore, we will test the hypothesis that the host elaborates cell wall-targeting defenses against bacteria in the cytosol through a forward genetic screen and parallel untargeted proteomics approach. Preliminary phosphoproteomic and genetic suppressor analyses of wild-type and ?prkA Lmo during ?-lactam exposure in vitro revealed that PrkA phosphorylates ~50 proteins, including the broadly conserved protein of unknown function IreB, during cell wall stress. However, the importance of IreB and other putative PrkA substrates in the context of the cytosol remains unknown. In this proposal, I will elucidate the function of IreB, assess its role in Lmo cytosolic survival, and test for functional conservation in other related organisms. To comprehensively assess the role of PrkA in cytosolic stress responses, I will execute parallel ex vivo phosphoproteomic analysis and in vivo suppressor selection to identify PrkA targets relevant during infection. To identify CADs that kill non-adapted bacteria in the cytosol, I will use a genome-wide CRISPR/Cas9 mutagenesis screen and proteomic analysis of Lmo isolated from the cytosol of macrophages to identify host factors that kill non-cytosol-adapted bacteria. Putative CADs will be validated through standard genetic approaches, and initial functional characterization will be performed as necessary. Completion of the Aims herein will provide insights into bacterial adaptations to cytosolic cell wall stress and will identify host factors that kill maladapted bacteria in this compartment. These studies therefore have the potential to illuminate new avenues for both pathogen- and host-directed therapies to combat antibiotic-resistant bacteria.